Thermostable variants of T7 RNA polymerase

Abstract

A bacteriophage RNA polymerase variant is provided. In some embodiments, the variant may have increased thermostability relative to the corresponding wild type bacteriophage RNA polymerase and/or wild type T7 RNA polymerase. Compositions, kits and methods that employ the variant are also provided.

What is claimed is: 
     
       1. An RNA polymerase, wherein the RNA polymerase:
 (i) has at least 90% sequence identity to SEQ ID NO: 1; and 
 (ii) comprises an amino acid substitution at the position corresponding to position 388 and at the position corresponding to position 567 of SEQ ID NO: 1. 
 
     
     
       2. The RNA polymerase of  claim 1 , wherein the RNA polymerase further comprises an amino acid substitution of at least one position corresponding to positions selected from 109, 205, 534, and 618 of SEQ ID NO:1. 
     
     
       3. The RNA polymerase of  claim 1 , wherein the RNA polymerase further comprises an amino acid substitution of at least two positions corresponding to positions selected from 109, 205, 534, and 618 of SEQ ID NO:1. 
     
     
       4. The RNA polymerase of  claim 1 , wherein the RNA polymerase further comprises an amino acid substitution at positions corresponding to 109, 205, 534, and 618 of SEQ ID NO:1. 
     
     
       5. The RNA polymerase of  claim 1 , wherein the RNA polymerase comprises one or more of the following amino acids substitutions selected from I109L, H205S, D388E, L534V, V567P and G618Q wherein the amino acid substitutions are at positions that correspond to positions in SEQ ID NO:1. 
     
     
       6. The RNA polymerase of  claim 1 , wherein the RNA polymerase further comprises an amino acid substitution at one or more positions corresponding to positions selected from: 75, 83, 108, 206, 227, 281, 297, 312, 323, 327, 333, 340, 354, 362, 375, 428, 446, 454, 461, 495, 510, 584, 591, 642, 711, 724, 740, 788, 832, 834, 835, 843, 847, 849, 856, 863, 866 and 877 of SEQ ID NO:1. 
     
     
       7. The RNA polymerase of  claim 1 , wherein the RNA polymerase further comprises an amino acid substitution of at least 10 positions corresponding to positions selected from: 75, 83, 108, 206, 227, 281, 297, 312, 323, 327, 333, 340, 354, 362, 375, 428, 446, 454, 461, 495, 510, 584, 591, 642, 711, 724, 740, 788, 832, 834, 835, 843, 847, 849, 856, 863, 866 and 877 of SEQ ID NO:1. 
     
     
       8. The RNA polymerase of  claim 1 , wherein the RNA polymerase further comprises one or more of the following amino acids substitutions selected from: T75Q, A83K, E108L, K206P, V227I, I281P, V297I, Y312D, A323I, A327P, K333P, V340E, A354Q, M362P, T375K, T375N, A428P, L446F, K454P, K461R, S495N, C510Q, A584K, D591E, K642R, K711R, A724P, K740R, G788A, M832F, D834E, T835L, A843Q, D847E, F849V, S856T, A863P, A866K and E877R, wherein the amino acid substitutions are at positions that correspond to positions in SEQ ID NO:1. 
     
     
       9. The RNA polymerase of  claim 1 , wherein the RNA polymerase further comprises at least 10 of the following amino acids substitutions selected from: T75Q, A83K, E108L, K206P, V227I, I281P, V297I, Y312D, A323I, A327P, K333P, V340E, A354Q, M362P, T375K, T375N, A428P, L446F, K454P, K461R, S495N, C510Q, A584K, D591E, K642R, K711R, A724P, K740R, G788A, M832F, D834E, T835L, A843Q, D847E, F849V, S856T, A863P, A866K and E877R wherein the amino acid substitutions are at positions that correspond to positions in SEQ ID NO:1. 
     
     
       10. The RNA polymerase of  claim 1 , wherein the RNA polymerase comprises a fusion to an exogenous DNA binding domain. 
     
     
       11. The RNA polymerase of  claim 1 , wherein, as a result of the amino acid substitutions, the RNA polymerase has increased stability at temperatures above 45° C., 50° C. or 55° C. relative to the T7 RNA polymerase of SEQ ID NO:1. 
     
     
       12. The RNA polymerase of  claim 1 , wherein, as a result of the amino acid substitutions, the RNA polymerase has increased activity at temperatures above 42° C., 45° C., 50° C. or 55° C. relative to the T7 RNA polymerase of SEQ ID NO:1. 
     
     
       13. The RNA polymerase of  claim 1 , wherein the polymerase has at least 95% sequence identity to SEQ ID NO:1. 
     
     
       14. The RNA polymerase of  claim 1 , wherein the polymerase has at least 95% sequence identity to any of SEQ ID NO:52-70. 
     
     
       15. A composition comprising:
 (i) the RNA polymerase of  claim 1 ; and 
 (ii) a buffering agent. 
 
     
     
       16. The composition of  claim 15 , further comprising ribonucleoside triphosphates and/or a modified nucleotide. 
     
     
       17. The composition of  claim 15 , further comprising a template DNA molecule comprising: a bacteriophage RNA polymerase promoter, operably linked to a target nucleotide sequence to be transcribed. 
     
     
       18. A kit comprising:
 (i) the RNA polymerase of  claim 1 ; and 
 (ii) a reaction buffer. 
 
     
     
       19. The kit of  claim 18 , wherein the kit further comprises one or more ribonucleoside triphosphates. 
     
     
       20. A method for synthesizing an RNA molecule comprising:
 (a) combining the RNA polymerase of  claim 1 , with ribonucleoside triphosphates that optionally comprise a modified ribonucleotide and a template DNA molecule comprising a bacteriophage RNA polymerase promoter that is operably linked to a target nucleotide sequence to be transcribed, to produce a reaction mix; and 
 (b) incubating the reaction mix to transcribe the template DNA molecule into RNA. 
 
     
     
       21. A method for synthesizing an RNA molecule comprising:
 incubating a reaction mix comprising the RNA polymerase of  claim 1 , ribonucleoside triphosphates optionally including one or more modified ribonucleotides, and a template DNA molecule comprising a bacteriophage RNA polymerase promoter that is operably linked to a target nucleotide sequence to be transcribed, thereby transcribing the template DNA molecule into RNA. 
 
     
     
       22. The method of  claim 20 , wherein the incubating is done at a temperature of at least 45° C. 
     
     
       23. The method of  claim 20 , wherein the bacteriophage RNA polymerase promoter is a T7 RNA polymerase promoter. 
     
     
       24. The method of  claim 21 , wherein the method is a Nucleic acid sequence-based amplification (NASBA) method that comprises: reverse transcribing an RNA template to produce an RNA-cDNA hybrid, treating the RNA-cDNA hybrid to destroy the RNA template and produce a DNA strand, hybridizing a primer to the DNA strand, wherein the primer comprises the bacteriophage RNA polymerase promoter, extending the primer to produce a second strand, and transcribing the second strand using the RNA polymerase to produce an RNA product. 
     
     
       25. The method of  claim 21 , further comprising detecting the RNA product.